The present invention is directed to proteoliposomes, their construction and use. Preferably the proteoliposome contains an integral membrane protein, having at least one transmembrane domain.
Advances in genomics have resulted in the discovery and identification of numerous proteins. These advances have made it possible to obtain transcripts and DNA encoding a range of proteins, including putative integral membrane proteins having multiple transmembrane domains, as well as the proteins themselves. The availability of such proteins makes it possible to identify ligands that interact with these proteins, permitting one to better understand the biology of these proteins and/or screen for compounds that modulate the function of such proteins. However, there are increasing problems in knowing what a specific protein actually does and/or finding simple and accurate methods that actually identify the ligands that interact with a particular protein. For example, a protein such as a receptor protein for which a ligand has not yet been identified is referred to as an xe2x80x9corphan proteinxe2x80x9d. Such orphan proteins are becoming more numerous as more DNA sequences, including DNA sequences encoding putative receptors, become available. In these cases, the DNA and proteins are classified based upon homologies to known proteins. For example, one can recognize conserved sequences that resemble a known domain, such as a transmembrane domain, thus indicating that the identified protein resides in a membrane (i.e., is an integral membrane protein).
Transmembrane proteins or integral membrane proteins are amphipathic, having hydrophobic domains that pass through the membrane and interact with the hydrophobic lipid molecules in the interior of the bilayer, and hydrophilic domains which are exposed to the aqueous environment on both sides of the membrane (for example, the aqueous environments inside and outside of the cell). The biological activities of integral membrane proteins (e.g., ligand binding) are dependent upon the hydrophilic domains; in some cases, the membranexe2x80x94spanning regions contribute to function.
Despite our ability to predict extra-membrane protein regions with some confidence, the prediction of the actual structure of these regions and the ligands bound thereto is much more tenuous. For the most part, the identification of natural and unnatural ligands of integral membrane proteins is an empirical process.
The identification of ligands and the study of their binding properties is more complicated for integral membrane proteins than for water-soluble proteins. Water-soluble proteins can be readily purified in aqueous buffers and maintained in a native conformation under such circumstances. Integral membrane proteins cannot be solubilized in aqueous buffers but must be maintained in an environment that allows the membrane-spanning region to maintain hydrophobic contacts. This is most often accomplished by including detergents in the solubilization buffer. When mixed with integral membrane proteins, the hydrophobic regions of the detergent bind the transmembrane region of the protein, displacing the lipid molecules of the membrane.
Although solubilizing transmembrane proteins in detergents in theory allows their purification, in practice, it is typically difficult to effectively isolate that protein from other membrane proteins while retaining native conformation for extended periods of time. For example, the calcium pump from the sarcoplasmic reticulum can only be isolated with its native structure intact when maintained within the context of the sarcoplasmic reticular membrane (Zhang et al. (1998), Nature 392: 835-39). Similarly, a three-dimensional map of the plasma membrane H+-ATPase was only possible when two-dimensional crystals were grown directly on electron microscope grids (Auer et al. (1998), Nature 392: 840-3). For many other transmembrane proteins, including the cystic fibrosis transmembrane conductance regulator (CFTR), it has not yet been possible to purify the protein for extended periods of time while maintaining the wild-type conformation.
Additionally, identifying the actual ligands that interact with such a transmembrane protein, while extremely important, has many difficulties. For example, the transmembrane protein needs to be in the proper conformation in order to interact with ligands. Yet part of the way that transmembrane proteins maintain their conformation is by being part of a cellular membrane. The current solutions to this problem are less than optimal. For some integral membrane proteins that span the membrane only once, the extracellular and/or intracellular domains can be synthesized as independent entities and, in some cases, will fold properly. However, this is not always true. Furthermore, the post-translational modifications made to soluble versions of the extracellular or intracellular domains often differ from those of the full-length membrane-bound protein. These differences can exert profound effects on ligand binding or other functional properties. For the vast majority of integral membrane proteins, which span the membrane more than once, even this less-than-ideal solution is not feasible. Typically, cell-based screens are utilized to identify ligands of interest with these proteins. Cell lines that express the integral membrane protein of interest are established and compared to a parental cell line not expressing the protein. However, in such cases, it is difficult to effectively isolate the protein of interest from other proteins that are also present in the cell membrane. In many cases, the protein of interest is expressed in lower amounts than other integral membrane proteins. Thus, there can be interference caused by a compound or ligand interacting with an entirely different protein. For phenotypic screens, it may be that one protein is involved in one stage of a large pathway involving multiple proteins. In such cases, the readout in the screening assay may be affected even when the protein of interest is not directly affected. Accordingly, it would be desirable to have a method to look at a specific integral membrane protein in its native confirmation where it can be isolated from other competing proteins.
Seven-transmembrane segment, G protein-coupled receptors (GPCRs) represent approximately 1-2 percent of the total proteins encoded by the human genome and are important targets for pharmaceutical intervention. GPCRs have seven transmembrane domains, and also include chemokine receptors such as CCR5 and CXCR4, which have been identified as cofactors in permitting the human immunodeficiency virus (HIV) to enter cells. Generally low levels of expression and the dependence of the native conformation of GPCRs on the hydrophobic, intramembrane environment have complicated the study of these proteins. Analysis of ligand interactions with GPCRs and screening for inhibitors of such interactions are commonly conducted using live cells or intact cell membranes. Typically, the binding of radiolabeled ligand with the cells or the induction of intracellular calcium levels by the ligand are used as readouts in such screens. A significant drawback of such assays are the extremely large number of cells required for high-throughput screening. Furthermore, such studies can be complicated by the presence of numerous cell surface proteins, many of which are expressed at much higher levels than the GPCR of interest. Thus, certain approaches, such as using the GPCR-expressing cells to identify either natural or synthetic ligands in a complex mixture, are precluded. In addition, the generation of monospecific antibodies directed towards a particular GPCR in the complex cell membrane environment is inefficient. Furthermore, for some GPCRs, like the chemokine receptors, multiple ligands bind a single receptor, and conversely, a single ligand can bind multiple receptors. Therefore, if the cell expresses more than one receptor for the ligand being studied, interpretation of the results can be complicated.
Traditional methods of synthesizing and isolating a recombinant protein and then testing it in various assays have proven difficult with integral membrane proteins having multiple transmembrane spanning domains because they do not typically retain wild-type conformation under standard conditions for extended periods of time. For example, Bieri et al. (1997), Nature Biotechnology 17:1105-1108, used a sensor chip covered with a mixed self-assembled lipid monolayer to stabilize the G protein-coupled receptor (GPCR), rhodopsin. However, unlike most GPCRs, rhodopsin can be purified easily and its function is well-known. Moreover, it is expressed at high levels and is not denatured as readily in harsh detergents as other GPCRs. Even so, the protein was only stable for a number of hours. Further, the method used to detect the ligand it interacts with, surface plasmon resonance (SPR), looks at uncoupling of the ligands via changes in molecular weight of the ligand-receptor complex. If the ligand is roughly equivalent in mass to the G proteins, the loss of coupled G protein induced by the binding of a protein ligand would result in little overall change in the mass of the complex, and not be detected. Thus, for most GPCRs, establishing cell-free systems for screening for agonistic and antagonistic ligands remains an elusive goal.
Accordingly, it would be desirable to produce and isolate in purified form these multiple transmembrane domain proteins while retaining their wild-type conformation. It would be desirable if these proteins could be maintained in their wild-type conformation for extended periods of time and under conditions commonly found in vivo. It would also be desirable if this could be applied to a wide range of integral membrane proteins. The purification of integral membrane proteins, particularly those that span the membrane more than once, in a functionally relevant conformation should expedite the search for ligands, both natural and unnatural, that bind to these proteins.
We have now discovered a method for expressing integral membrane proteins in large amounts, purifying and isolating them from other proteins, while maintaining them in a wild-type conformation for extended periods of time.
Preferably, the integral membrane protein (sometimes referred to as a transmembrane protein) has a plurality of transmembrane domains. The known integral membrane proteins may cross the membrane only once or, for example, up to 16 times. One simple way to classify these proteins is by the number of transmembrane domains (Table 1, infra). Preferred proteins include G protein-coupled receptors (GPCRs), ion channels, amino acid transporters, glucose transporters, phosphate transporters, CFTR, and nuclear receptor complex proteins. Preferably, the proteins are eukaryotic, bacterial or viral membrane proteins; still more preferably the proteins are mammalian membrane proteins.
The desired protein is extended by a short peptide epitope tag, for example the C9 tag, which can be recognized by an antibody (for example, the 1D4 antibody). The tag can be added to the N-terminus or to the C-terminus of the protein, depending upon the ultimate orientation of the protein in the proteoliposome that is desired. The desired protein is expressed in a cell. Codon optimization may be used to increase the expression level of the protein. The protein is then isolated from the cell by a solubilizing agent that maintains the protein""s conformation. Preferably, the solubilizing agent is a detergent. Preferred detergents include alkyl glucopyranosides (such as C8CP, C10-M, C12-M, Cymal-5, Cymal-6 and Cymal-7), alkyl sucroses (such as HECAMEG), digitonin, CHAPSO, hydroxyethylglucamides (such as HEGA-10), oligoethyleneglycol derivatives (such as C8ES, C8En and C12E8), dodecylmaltopyranoside, and phenyl polyoxethylenes (such as Triton X-100).
The detergent-solubilized protein is then separated from the other cellular debris by capture onto a solid surface (e.g. a spherical or elliptoid bead). The bead has on its surface an antibody or other specific ligand that will capture, orient and concentrate the protein on the surface of the bead. This isolated protein is maintained in its wild-type conformation. Thereafter, it is mixed with a lipid component. One may also add an attractant for the lipid on the bead surface. For example, the bead can be streptavidin-coated and some lipid component (e.g. biotinyl-DPPE) can be covalently conjugated to biotin. The bead with the mixture is then subjected to a known means such as dialysis to form the proteoliposome. The streptavidin-biotin interaction, in this example, helps to attach the lipid layer to the bead surface as the detergent is removed. The resulting proteoliposome will maintain the integral membrane protein in its native conformation in an isolated and/or purified form for extended periods of times.
These proteoliposomes can be used as immunogens to elicit immune reactions. Alternatively, they can be used to screen antibody libraries for an antibody.
In a preferred embodiment, the stable proteoliposomes can be used as antigens to screen antibody libraries, including phage display antibody libraries.
These proteoliposomes can also be used in screening assays such as drug screening and identifying ligands.
These proteoliposomes can also be used to determine the protein""s structure.
These proteoliposomes can also be used as a vaccine.